The present invention is related to genetic engineering of plants. The invention is particularly related to the transformation of plants using recombinant DNA techniques to amplify a gene of interest and express a protein of interest.
A predominant mode of plant transformation employs A. tumefaciens, in which a transforming DNA (T-DNA) is modified to incorporate a desired foreign gene. The recombinant T-DNA contains the desired foreign gene between flanking non-coding regulatory sequences and the left and right border regions of the wild-type tumor-inducing (Ti) plasmid. The recombinant T-DNA can be provided as part of an integrative plasmid, which integrates into a wild-type Ti plasmid by homologous recombination. Typically, however, the recombinant T-DNA is provided in a binary vector and transferred into a plant cell through the action of trans-acting vir genes on a helper Ti plasmid. The T-DNA integrates randomly into the nuclear genome with some of the transformants permitting expression of the desired protein (Zambryski, 1988). Because of the random integration event of the T-DNA into the nuclear chromosome, variability of transcription level is expected, and transformants are screened to identify those showing the highest levels of foreign gene expression. Those transformants expressing the highest levels of foreign protein can be propagated and multiplied in tissue culture before transplanting to soil.
Many plant species previously recalcitrant to gene transfer are now amenable, including cereal crops (McElroy et al., 1994). Plants have the capacity to express foreign genes from a wide range of sources, including viral, bacterial, fungal, insect, animal, and other plant species. In single-copy nuclear transgenics, foreign protein in excess of 1% of total protein is often achieved (Hiatt et al., 1989). Further, assembly and processing of complex animal proteins in plants is possible, e.g., human serum albumin (Sijmons et al., 1990) and secretory antibodies (Ma et al., 1995a). Recently, expression of correctly processed avidin was reported in corn seed at a level of 2% of the total soluble protein (Hood et al., 1997). It has been estimated that the cost of recombinant protein production in plants (assuming the foreign protein is 10% of total protein) can be 10 to 50 times less than in E. coli by fermentation (Kusnadi et al., 1997).
Plants have been used as expression systems for vaccine antigens (Mason et al., 1995). The expression of vaccine antigens in tobacco plants has been reported and the plant material has been shown to be orally immunogenic in mice (work reviewed by Mason et al., 1995; Arntzen et al., 1996). Complex antibodies have also been expressed in plants, which correctly processed and assembled the antibody chains into IgG and secretory IgA forms (review by Ma et al., 1995b). In the latter case, four different genes were coordinately expressed, including the IgA heavy and light chains, the joining component, and the secretory component, which faithfully assembled in plant cells. Further, expression and accumulation of antibodies in corn and soybean seeds has been reported.
However, a major limitation in the use of plants for expression and delivery of a protein of interest is the rather low level of expression usually obtained, which ranges from 0.01% to 2% of the total soluble protein. For example, soybeans contain 40% protein by weight, yet current methods for foreign protein expression yield no more than 2% of the total protein in seeds. Synthetically produced recombinant vaccine proteins, which avoid the hazards associated with using live or attenuated virus, can be produced in cell culture systems, e.g., hepatitis B surface antigen (Cregg et al., 1987) and dengue virus proteins (Sugrue et al., 1997). However, the cost of cell culture systems is often so high as to preclude vaccination on a large scale, particularly in poor countries.
In applications requiring overexpression of a purified protein of interest, high-level expression would greatly facilitate the purification process. Therefore, a method of amplifying a gene of interest and overproducing a protein of interest in recombinant plants is desired.
Previous techniques are, however, inherently self-limiting by virtue of xe2x80x9csuccessfulxe2x80x9d transformation affording only one or a few functional copies of the foreign gene integrated into the plant genome. Efforts to increase the level of expression under such circumstances are therefore limited to optimizing the promoter and/or enhancer sequences, using synthetic versions of the foreign gene optimized for expression in the plant host, optimizing the termination sequence, optimizing expression of transcription factors, and the like. These measures can be expected to enhance expression of the desired antigen, although such enhancement is still limited by the copy number of the foreign gene. True xe2x80x9camplificationxe2x80x9d of the foreign gene in plant cells, in which multiple functional copies of the gene are generated either extrachromosomally or integrated into the plant chromosome, is desired if much greater levels of protein expression are to be achieved. The geminiviruses are interesting candidates for producing marked amplification of transgenes in plants.
Members of the plant virus taxonomic family Geminiviridae are unique among viruses in possessing twinned or geminate virions. They are also unusual among plant viruses in that they possess single-stranded circular DNA genomes. The three genera of Geminiviridae are: the leafhopper-transmitted Mastreviruses (type member: maize streak virus, MSV); the leaf- and planthopper-transmitted Curtoviruses (type member: beet curly top virus, BCTV); and the whitefly-transmitted Begomoviruses (type member: bean golden mosaic virus, BGMV). Until recently, the three genera were known as Subgroups I, II and III, respectively. Mastreviruses and Curtoviruses have only a single genomic component of approximately 2.5 to 2.8 kb; Begomoviruses may have one or two components of the same size, one of which is dependent on the other for replication. Mastreviruses have the simplest organization, with Curtoviruses and Begomoviruses sharing a very similar and more complex organization. An overview of the genetic organization of geminivirus genomes is shown in FIG. 1.
The geminiviruses replicate via a rolling circle mechanism, analogous to that used by phage "PHgr"X174 and ssDNA plasmids of gram positive microorganisms. The only exogenous a protein required for replication is the viral replication initiation (Rep) protein encoded by a geminiviral replicase gene. This multifunctional protein initiates replication at a conserved stem loop structure found in the viral origin of replication by inducing a nick within a conserved nonanucleotide motif (TAATATTA↓C) found in the intergenic loop sequence. Transcription of the viral genome is bidirectional with transcription initially within the intergenic (IR) region. Rep also has functions involved in controlling the plant cell cycle, and possibly also in modulating the expression of host genes involved in DNA replication (reviewed by Palmer et al., 1997b). The Rep protein can act in trans, that is, it need not be expressed by the viral replicon itself, but can be supplied from another extrachromosomal viral replicon, or even from a nuclear transgene (Hanley-Bowdoin et al., 1990). The cis requirements for viral replication are the viral intergenic region/s (IR), which contain sequences essential for initiation of rolling circle replication (the long intergenic region (LIR) of Mastreviruses, or the intergenic region of other geminiviruses) and synthesis of the complementary strand (the short IR (SIR) of Mastreviruses).
Infectious clones of geminiviruses are commonly constructed as tandem dimers or partial dimers of the virus genome, usually with the origin of replication sequences duplicated. This facilitates escape of the cloned virus from the cloning vector sequences by a replicative release mechanism mediated by the Rep protein inducing a nick at each stem-loop structure and the host DNA replication machinery then displacing a ssDNA copy of the viral genome. This mechanism applies to rescue of replication-defective geminivirus genomes from chromosomally integrated partial multimers by the Rep protein of wild type virus (see, for example, Stanley et al., 1990). Moreover, the Rep protein can mediate replicative release of recombinant viral DNA integrated into the host cell chromosome (Hayes et al., 1988; 1989; Kanevski et al., 1992; Palmer, 1997; Palmer et al., 1997d).
Geminiviruses replicate to very high copy number in the nuclei of infected cells, via a double-stranded DNA replicative intermediate form (RF-DNA) and have therefore attracted interest for their potential use in gene amplification strategies to increase the copy number and enhance the expression levels of foreign genes linked to the viral replicon (reviewed by Palmer et al., 1997a and Timmermans et al., 1994). Geminiviruses seem to be fairly plastic with respect to the size of foreign DNA that can be linked to the viral replicon without inhibiting DNA replication. There is, however, a stringent size limitation imposed on movement of virus genomes. This issue becomes irrelevant, however, if the geminiviral replicon is used in a transgene amplification system where a partial dimer of a xe2x80x9cmaster copyxe2x80x9d of the viral replicon (lacking the genes involved in viral movement) is integrated into every cell of a transgenic plant (Palmer et al., 1997a). The circular monomeric viral replicon is then mobilized from the master copy in the chromosome by areplicative release mechanism (Stenger et al., 1991), and the recombinant viral vector replicates to very high copy number as a nuclear episome (e.g. Hayes et al. ,1988; 1989; Kanevski etal., 1992; Palmer et al., 1997d). The RF-DNA forms of geminiviral genomes exist as histone-associated minichromosome structures, and their genes are transcribed by the host RNA polymerase complex. Transcription of genes linked to a geminiviral replicon should therefore be regulated by their respective promoter sequences.
U.S. Pat. Nos. 5,589,379 and 5,650,303, issued to Kridl et al., disclose use of a geminiviral gene transfer vector to permit inducible expression of a foreign gene in plants. In this approach, a first expression cassette has a foreign protein coding sequence under the control of the coat protein promoter native to the geminivirus. A second expression cassette has a geminiviral trans-acting transcription factor under the control of a plant-inducible promoter. In this way, transcription of the foreign gene reportedly can be regulated by the inducible expression of the trans-acting transcription factor.
A gene amplification approach to increasing the copy number and expression of recombinant transgenes could dramatically increase the level of desired foreign proteins in plants. This could lead to safer and more economical production of a protein of interest. In this regard, a transgene vector system that utilizes the release and replication capabilities of geminiviruses is particularly worthy of investigation.
Needham et al. disclose a binary vector containing the following elements derived from tobacco yellow dwarf virus: two copies of an LIR flanking an SIR and two complementary sense open reading frames (C1 and C2) which contain an intron, which when processed, produces a Rep protein under the transcriptional control of its native promoter within the LIR. This vector does not include open reading frames encoding the putative movement (V1) and coat (V2) proteins. Needham et al. disclose further that transgenic tobacco plants transformed with this vector further comprising a reporter gene, demonstrate release and episomal replication of the viral elements of the vector, and expression of the reporter gene (Needham et al., 1998, Plant Cell Reports, 17:631)
Atkinson et al. disclose an episomal vector containing the following elements derived from tobacco yellow dwarf virus: two copies of an LIR flanking both an SIR and two complementary sense open reading frames, C1 and C2 that produce Rep. The expression of the Rep protein is under the transcriptional control of its native promoter within the LIR. Atkinson et al. also disclose that transgenic Petunia hybrida plants containing a CaMv 35S promoter-driven chalcone synthase A gene cloned into the episomal vector, demonstrate release and episomal replication of the viral elements of the vector and the chalcone synthase A gene (Ross et al., 1998, The Plant Journal, 15:593).
There is a need in the art for a method of increasing the copy number of a recombinant transgene in a plant.
There is also a need in the art for a method of increasing the level of expression of a recombinant transgene in a plant.
There is also a need in the art for a method of increasing the level of protein expressed from a recombinant transgene in a plant.
The invention provides a pair of recombinant nucleic acid molecules wherein a first molecule comprises at least a portion of a long intergenic region (LIR) of a geminivirus genome and wherein the first molecule lacks a functional geminiviral coat protein encoding sequence, and a second molecule comprising a geminiviral replicase gene operably linked to a fruit ripening-dependent promoter.
As used herein, a xe2x80x9clong intergenic regionxe2x80x9d (LIR) refers to a noncoding region that contains sequences capable of forming a hairpin structure, including a conserved 9-base sequence (TAATATTA↓C) found in all geminiviruses.
As used herein, xe2x80x9cat least a portion of a long intergenic regionxe2x80x9d refers to a region of a long intergenic region (LIR) that contains a rep binding site capable of mediating excision and replication by a geminivirus Rep protein. The LIR of the geminivirus, bean yellow dwarf virus is 303 nucleotides (FIG. 2). As used herein, xe2x80x9cat least a portion of a long intergenic repeatxe2x80x9d refers to a fragment of the long intergenic repeat that is less than 303 nucleotides. xe2x80x9cAt least a portionxe2x80x9d of a long intergenic region encompasses for example 50 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 264 nucleotides, 270 nucleotides, 275 nucleotides, 280 nucleotides, 285 nucleotides, 290 nucleotides or 300 nucleotides. An LIR according to the invention includes an LIR of mastrevirus as well as an intergenic region (IR) of either curtovirms or begomovirus.
As used herein, xe2x80x9cfruit ripening dependentxe2x80x9d refers to inducible under fruit ripening conditions and/or expressed in a tissue specific manner.
By xe2x80x9ctissue specificxe2x80x9d is meant expressed only in tissues of the fruit or in leaves. Tissues of the fruit include vascular bundles, pericaip, collumella, epidermis, placental tissue, locular tissue and seeds. As used herein, xe2x80x9ctissue specificxe2x80x9d also refers to expressed in a seed specific manner.
As used herein, xe2x80x9cseed specificxe2x80x9d refers to expressed only in the developing seed encompassing the cotyledon and the embryo axis, but not including the root and the stem. As used herein, xe2x80x9cseed specificxe2x80x9d also refers to expressed in the endosperm and perisperm.
A xe2x80x9cfruit ripening-dependent promoterxe2x80x9d according to the invention does not include a native viral Rep protein promoter included in an LIR. The LIR sequence of Bean yellow dwarf 30 virus, including the putative TATA box (TTATA, boxed in FIG. 2) is presented in FIG. 2, for example. Sequences of other geminivirus LIRs are available in the literature. As used herein, the native rep gene promoter is located in the LIR and can regulate rep gene expression in a phloem specific manner. Therefore, the invention does not encompass control of the rep gene by a native rep promoter, which native promoter is found in an LIR (FIG. 2), and includes the TATA sequence TTATA (boxed in FIG. 2).
As used herein, a xe2x80x9cfruit ripening-dependent promoterxe2x80x9d can also be expressed in phloem wherein it demonstrates development-stage dependent expression encompassing expression during fruit ripening or embryo storage protein deposition.
As used herein, xe2x80x9cdevelopment stagexe2x80x9d refers to a particular period of cell growth, differentiation, and organization of cells which can be characterized by changes in anatomy, biochemistry, or the coordinate expression of a particular set of genes.
As used herein, xe2x80x9cdevelopingxe2x80x9d refers to the process of growth, differentiation and organization of cells that occurs during the formation of a tissue (e.g. epidermis, phloem, xylem, parenchyma etc . . . ) or an organ (e.g. leaf, stem, root, flower, fruit or seed).
As used herein, xe2x80x9cfruitxe2x80x9d refers to the ovary of an angiosperm flower and the associated structures (e.g. the receptacle or parts of the floral tube) that enlarge and develop to form a mass of tissue surrounding the seeds. According to the invention, the particular tissues that are involved in fruit development vary with the species, but tissues involved in fruit development according to the invention, are always derived from the maternal parent of the progeny seeds.
As used herein, xe2x80x9cripexe2x80x9d refers to a stage of fruit development that is characterized by changes in pigmnentation, the conversion of acids and starches to free sugars, and breakdown of cell walls that results in softening of the fruit.
As used herein, xe2x80x9cfruit ripening conditionsxe2x80x9d refer to conditions under which the developmental processes involved in fruit ripening can occur, including cell division and expansion of maternal tissues that occurs after fertilization of ovaries. As used herein, for example, production of ethylene is a chemical signal that stimulates the genetic program for ripening in climacteric fruits such as tomato.
As used herein, xe2x80x9cembryo storage protein depositionxe2x80x9d refers to the synthesis and accumulation of storage proteins in the parts of the embryo, particularly cotyledons, or seeds of species that lack endosperm and in which the embryo is large and contains most of the seed storage tissue (including for example, Leguminosae, Cucurbitaceae, Compositaea, Solanaceae, Brassicaceae).
xe2x80x9cinduciblexe2x80x9d refers to expressed in the presence of an exogenous or endogenous chemical (for example an alcohol, a hormone, or a growth factor), in the presence of light and/or in response to developmental changes.
As used herein, xe2x80x9cendogenousxe2x80x9d refers to naturally occurring in a plant.
As used herein, xe2x80x9cexogenousxe2x80x9d refers to not naturally occurring in a plant.
As used herein, xe2x80x9cinduciblexe2x80x9d also refers to expressed in any tissue in the presence of a chemical inducerxe2x80x9d. As used herein, xe2x80x9cchemical inductionxe2x80x9d according to the invention refers to the physical application of a exogenous or endogenous substance (including macromolecules e.g. proteins, or nucleic acids) to a plant or a plant organ (e.g. by spraying a liquid solution comprising a chemical inducer on leaves, application of a liquid solution to roots or exposing plants or plant organs to gas or vapor) which has the effect of causing the target promoter present in the cells of the plant or plant organ to increase the rate of transcription.
As used herein, xe2x80x9cprotein of interestxe2x80x9d refers to any protein that is either heterologous or endogenous to a geminivirus or plant.
As used herein, xe2x80x9cgene of interestxe2x80x9d refers to any gene that is either heterologous or endogenous to a geminivirus or plant.
As used herein, xe2x80x9cnucleotide sequence of interestxe2x80x9d refers to any nucleotide sequence that is either heterologous or endogenous to a geminivirus or plant. A nucleotide sequence of interest refers to DNA or RNA.
xe2x80x9cHeterologousxe2x80x9d refers to a gene which is not naturally present in a geminivirus genome or a gene which is not naturally present in a plant genome. xe2x80x9cHeterologousxe2x80x9d also refers to a protein which is not naturally expressed from a geminivirus genome or a plant genome.
xe2x80x9cEndogenousxe2x80x9d refers to a gene which is naturally present in a geminivirus genome or a plant genome. xe2x80x9cEndogenousxe2x80x9d also refers to a protein which is naturally expressed from a geminivirus genome or a plant genome.
As used herein, the term xe2x80x9coperably linkedxe2x80x9d refers to the respective coding sequence being fused in-frame to a promoter, enhancer, termination sequence, and the like, so that the coding sequence is faithfully transcribed, spliced, and translated, and the other structural features are able to perform their respective functions.
In a preferred embodiment, the first molecule further comprises an SIR.
As used herein, xe2x80x9cSIRxe2x80x9d refers to a noncoding region of a Mastrevirus genome containing the putative complementary strand origin of replication, the binding site for a short DNA primer that primes synthesis of the complementary DNA strand and consensus polyadenylation signals in both strands. As used herein, xe2x80x9cSIRxe2x80x9d refers to a region of DNA that is approximately 150 base pairs and extends from the termination codon of the geminivirus coat proteins (V2) to the termination codon of one of the open reading frames encoding the Rep protein (C2).
According to the invention, a gene of interest can be located either 5xe2x80x2 of an SIR or 3xe2x80x2 of an SIR in a recombinant nucleic acid molecule.
In another preferred embodiment, the first molecule further comprises a plant-functional promoter.
In another preferred embodiment, the plant-fulnctional promoter is selected from the group consisting of CaMV 35S, tomato E8, patatin, ubiquitin, mannopine synthase (mas), rice actin 1, soybean seed protein glycinin (Gy1) and soybean vegetative storage protein (vsp).
In another preferred embodiment, the first molecule further comprises a gene of interest.
In another preferred embodiment, the gene of interest is a heterologous gene.
In another preferred embodiment, the gene of interest of the first molecule is selected from the group consisting of a gene encoding luciferase, glucuronosidase (GUS), green fluorescent protein (GFP), shigatoxin B (StxB), staphylococcus enterotoxin B (SEB), E. coli labile toxin B (LT-B), Norwalk virus capsid protein (NVCP), and hepatitis B surface antigen (HBsAg).
In another preferred embodiment, the first molecule further comprises a plant-functional termination sequence.
In another preferred embodiment, the plant-functional termination sequence is selected from the group consisting of nopaline synthase (nos), vegetative storage protein (vsp), pin2, and geminiviral short intergenic (sir) termination sequences.
In another preferred embodiment, the nucleotide sequence of the first DNA molecule is optimized for expression in plants by having at least one codon degenerate to a corresponding codon of the native protein encoding sequence.
In another preferred embodiment, the first molecule is single stranded.
The invention also provides a recombinant nucleic acid molecule comprising at least a portion of a long intergenic region (LIR) of a geminivirus genome and a geminiviral replicase gene operably linked to a fruit ripening-dependent promoter.
In a preferred embodiment, the recombinant nucleic acid molecule further comprises an SIR.
In another preferred embodiment, the recombinant nucleic acid molecule further comprises a plant-functional promoter.
In another preferred embodiment, the plant-functional promoter is selected from the group consisting of CaMV 35S, tomato E8, patatin, ubiquitin, mannopine synthase (mas), rice actin 1, soybean seed protein glycinin (Gy1) and soybean vegetative storage protein (vsp).
In another preferred embodiment, the recombinant nucleic acid molecule further comprises a gene of interest.
In another preferred embodiment, the gene is a heterologous gene.
In another preferred embodiment, the gene of interest is selected from the group consisting of a gene encoding luciferase, glucuronosidase (GUS), green fluorescent protein (GFP), shigatoxin B (StxB), staphylococcus enterotoxin B (SEB), E. coli labile toxin B (LT-B), Norwalk virus capsid protein (NVCP), and hepatitis B surface antigen (HbsAg).
In another preferred embodiment, the recombinant nucleic acid molecule further comprises a plant-functional termination sequence.
In another preferred embodiment, the plant-functional termination sequence is selected from the group consisting of nopaline synthase (nos), vegetative storage protein (vsp), pin2, and geminiviral short intergenic (sir) termination sequences.
In another preferred embodiment, the nucleotide sequence is optimized for expression in plants by having at least one codon degenerate to a corresponding codon of the native protein encoding sequence.
In another preferred embodiment, the recombinant nucleic acid molecule is single stranded.
The invention also provides an expression vector comprising a selectable marker gene and at least a portion of a long intergenic region (LIR) of a geminivirus genome, a restriction site for insertion of a gene of interest, a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter, and wherein said nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence.
In a preferred embodiment, the vector further comprises an SIR.
In another preferred embodiment, the vector lacks a functional geminiviral replicase gene.
In another preferred embodiment, the nucleotide sequence is flanked by two of said LIR portions.
In another preferred embodiment, the 5xe2x80x2 end of the nucleotide sequence is operably linked to a plant-functional promoter sequence.
In another preferred embodiment, the vector further comprises a gene of interest.
In another preferred embodiment, the gene is a heterologous gene.
In another preferred embodiment, the gene of interest is selected from the group consisting of a gene encoding a luciferase, glucuronosidase (GUS), green fluorescent protein (GFP), shigatoxin B (StxB), staphylococcus enterotoxin B (SEB), labile toxin B (LT-B), Norwalk virus capsid protein (NVCP), and hepatitis B surface antigen (HbsAg).
In another preferred embodiment, the 3xe2x80x2 end of the gene is operably linked to a plant-functional termination sequence.
In another preferred embodiment, the gene is optimized for expression in plants by having at least one codon degenerate to a corresponding codon of the native protein encoding sequence.
In another preferred embodiment, the vector further comprises an E. coli origin of replication.
In another preferred embodiment, the vector further comprises an Agrobacterium tumefaciens origin of replication.
In another preferred embodiment, the nucleotide sequence is flanked by left and right T-DNA border regions of Agrobacterium tumefaciens. 
The invention also provides for a strain of E. coli transfected with an expression vector comprising a selectable marker gene and at least a portion of a long intergenic region (LIR) of a geminivirus genome, a restriction site for insertion of a gene of interest, a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter, and wherein said nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence, and further comprising an E. coli origin of replication.
The invention also provides for a strain of Agrobacterium tumefaciens transfected with an expression vector comprising a selectable marker gene and at least a portion of a long intergenic region (LIR) of a geminiviras genome, a restriction site for insertion of a gene of interest, a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter, and wherein said nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence, and firther comprising an E. coli origin of replication, further comprising an Agrobacterium tumefaciens origin of replication.
In a preferred embodiment, the strain further comprises a helper tumor-inducing (Ti) plasmid.
The invention also provides for a transgenic plant cell transformed with a nucleic acid having at least a portion of a long intergenic region (LIR) of a gemninivirus genome, a gene of interest, wherein the nucleic acid lacks a functional geminiviral coat protein encoding sequence.
The invention also provides for a transgenic plant cell transformed with a nucleic acid comprising at least a portion of a long intergenic region (LIR) of a geminivirus genome flanking a restriction site for insertion of a gene of interest, and a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter and wherein the nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence.
In a preferred embodiment, the transgenic plant cell further comprises a heterologous gene.
In another preferred embodiment, the transgenic plant cell lacks a functional geminiviral replicase gene.
In another preferred embodiment, the nucleic acid is present in nuclear episomes in the cell.
In another preferred embodiment, the 5xe2x80x2 end of the gene of interest is operably linked to a plant-functional promoter sequence.
In another preferred embodiment, the gene of interest is selected from the group consisting of a gene encoding luciferase, glucuronosidase (GUS), green fluorescent protein (GFP), shigatoxin B (StxB), staphylococcus enterotoxin B (SEB), labile toxin B (LT-B), Norwalk virus capsid protein (NVCP), and hepatitis B surface antigen (HBsAg).
In another preferred embodiment, the 3xe2x80x2 end of the gene of interest is operably linked to a plant-functional termination sequence.
In another preferred embodiment, the gene of interest is optimized for expression in plants by having at least one codon degenerate to a corresponding codon of the native protein encoding sequence.
In another preferred embodiment, the transgenic plant cell further comprises a viral replicase encoding sequence operably linked to a plant functional promoter and a termination sequence.
In another preferred embodiment, transcription of the viral replicase encoding sequence is regulated by an inducible promoter.
In another preferred embodiment, the 5xe2x80x2 end of the viral replicase encoding sequence is operably linked to a tissue-specific promoter.
In another preferred embodiment, the tissue-specific promoter is selected from the group consisting of glucocorticoid, estrogen, jasmonic acid, insecticide RH5992, copper, tetracycline, and alcohol-inducible promoters.
In another preferred embodiment, the viral replicase encoding sequence encodes a wild-type geminiviral replicase.
In another preferred embodiment, the viral replicase encoding sequence is provided as an expression cassette or viral replicon.
The invention also provides for a transgenic plant seed transformed with a nucleic acid having at least a portion of a long intergenic region (LIR) of a geminivirus genome, a gene of interest, wherein the nucleic acid lacks a functional geminiviral coat protein encoding sequence.
The invention also provides for a transgenic plant seed transformed with a nucleic acid comprising at least a portion of a long intergenic region (LIR) of a geminivirus genome, a restriction site for insertion of a gene of interest, and a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter and wherein the nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence.
In a preferred embodiment, the seed further comprises a heterologous gene.
In another preferred embodiment, the nucleic acid lacks a functional geminiviral replicase gene.
In another preferred embodiment, the seed further comprises a viral replicase encoding sequence expressed in trans with the nucleotide sequence.
In another preferred embodiment, the 5xe2x80x2 end of the viral replicase encoding sequence is operably linked to a fruit ripening-dependent promoter.
In another preferred embodiment, the seed is selected from tobacco, tomato, potato, banana, soybean, pepper, wheat, rye, rice, spinach, carrot, maize and corn.
The invention also provides for a method of transforming a plant cell comprising contacting the plant cell with a strain of Agrobacterium tumefaciens transfected with an expression vector comprising a selectable marker gene and a nucleic acid sequence comprising at least a portion of a long intergenic region (LIR) of a geminivirus genome, a restriction site for insertion of a gene of interest, and a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter and wherein said nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence, and further comprising an Agrobacterium tumefaciens origin of replication, under conditions effective to transfer and integrate the nucleotide sequence into the nuclear genome of the cell.
In a preferred embodiment, the transformed plant cell is regenerated.
The invention also provides a method of transforming a plant cell comprising subjecting the plant cell to microparticle bombardment with solid particles loaded with a pair of recombinant nucleic acid molecules wherein a first molecule comprises at least a portion of a long intergenic region (LIR) of a geminivirus genome, and wherein the first molecule lacks a functional geminiviral coat protein encoding sequence, and a second molecule comprising a geminiviral replicase gene operably linked to a fruit ripening-dependent promoter, under conditions effective to transfer and integrate said nucleotide sequence into the nuclear genome of the cell.
The invention also provides a method of producing a transgenic plant comprising transforming a plant cell by a method comprising subjecting the plant cell to microparticle bombardment with solid particles loaded with a pair of recombinant nucleic acid molecules wherein a first molecule comprises at least a portion of a long intergenic region (LIR) of a geminivirus genome, and wherein the first molecule lacks a functional geminiviral coat protein encoding sequence, and a second molecule comprising a geminiviral replicase gene operably linked to a fruit ripening-dependent promoter under conditions effective to transfer and integrate said nucleotide sequence into the nuclear genome of the cell, and regenerating the plant cell.
The invention also provides a method of amplifying a heterologous nucleotide sequence in a transgenic plant comprising producing a transgenic plant by a method comprising transforming a plant cell by a method comprising subjecting the plant cell to microparticle bombardment with solid particles loaded with a pair of recombinant nucleic acid molecules wherein a first molecule comprises at least a portion of a long intergenic region (LIR) of a geminivirus genome, and wherein the first molecule lacks a functional geminiviral coat protein encoding sequence, and a second molecule comprising a geminiviral replicase gene operably linked to a fruit ripening-dependent promoter under conditions effective to transfer and integrate said nucleotide sequence into the nuclear genome of the cell, and regenerating the plant cell, and subjecting the transgenic plant to a wild-type geminivirus, which expresses a viral replicase in planta that rescues and replicates the nucleotide sequence in cells of the plant.
The invention also provides a method of overproducing a protein in a plant comprising producing a transgenic plant by the method of contacting the plant cell with a strain of Agrobacterium tumefaciens transfected with an expression vector comprising a selectable marker gene and a nucleic acid sequence comprising at least a portion of a long intergenic region (LIR) of a geminivirus genome, a restriction site for insertion of a gene of interest, and a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter and wherein said nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence, and further comprising an Agrobacterium tumefaciens origin of replication, under conditions effective to transfer and integrate the nucleotide sequence into the nuclear genome of the cell, ans subjecting the transgenic plant to a wild-type geminivirus, which expresses a viral replicase in planta that rescues and replicates the nucleotide sequence in said plant.
The invention also provides for a method of amplifying a heterologous nucleotide sequence in a transgenic plant comprising producing a transgenic plant by the method of contacting the plant cell with a strain of Agrobacterium tumefaciens transfected with an expression vector comprising a selectable marker gene and a nucleic acid sequence comprising at least a portion of a long intergenic region (LIR) of a geninivirus genome, a restriction site for insertion of a gene of interest, and a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter and wherein said nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence, and further comprising an Agrobacterium tumefaciens origin of replication, under conditions effective to transfer and integrate the nucleotide sequence into the nuclear genome of the cell, and subjecting the transgenic plant to a chemical or developmental agent, which induces expression of a viral replicase in planta that rescues and replicates the nucleotide sequence in the plant.
In a preferred embodiment, the inducible promoter is selected from the group consisting of glucocorticoid, estrogen, and alcohol-inducible promoters.
In another preferred embodiment, replication of the viral replicase is expressed in trans with the nucleotide sequence.
The invention also provides for a method of overproducing a protein in a plant, comprising producing a transgenic plant produced by the method comprising contacting the plant cell with a strain of Agrobacterium tumefaciens transfected with an expression vector comprising a selectable marker gene and a nucleic acid sequence comprising at least a portion of a long intergenic region (LIR) of a geminivirus genome, a restriction site for insertion of a gene of interest, and a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter and wherein said nucleic acid sequence lacks a functional geminiviral coat protein encoding sequence, and further comprising an Agrobacterium tumefaciens origin of replication, under conditions effective to transfer and integrate the nucleotide sequence into the nuclear genome of the cell, and subjecting the transgenic plant to a chemical or developmental agent, which induces expression of a viral replicase in planta that rescues and replicates the nucleotide sequence in the plant.
The invention also provides a recombinant nucleic acid molecule comprising a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter.
The invention also provides a vector comprising a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter.
The invention also provides a transgenic plant cell transformed with a nucleic acid comprising a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter.
In a preferred embodiment, the transgenic plant cell is selected from the group consisting of tobacco, tomato, potato, banana, soybean, pepper, wheat, rye, rice, spinach, carrot, maize and corn.
The invention also provides a transgenic plant seed transformed with a nucleic acid comprising a functional geminiviral replicase gene operably linked to a fruit ripening-dependent promoter.
In a preferred embodiment, the transgenic plant cell is selected from the group consisting of tobacco, tomato, potato, banana, soybean, pepper, wheat, rye, rice, spinach, carrot, maize and corn.
The present invention thereby affords a method of amplifying, i.e., increasing the copy number, of a desired nucleotide sequence, in the genome of a transgenic plant, thereby permitting expression of the encoded protein over basal levels obtained in the absence of amplification. Moreover, protein expression can be regulated in a fruit ripening-dependent manner.